It is generally recognized that circulating microvascular (endothelial) cells are important markers for a variety of disease states such as cardiac disease, cardiovascular disease and diabetes (Bos, C. J., et al., J. Am. Col. Cardiol. (2006) 48:1538-1547). Detecting such cells in the circulatory system is, however, problematic because of interference from other cells, such as platelets. Methods that involve separation of cells by type, or antibody labeling such as the use of antibodies directed to CD31, require time-consuming steps and often require expensive equipment.
PCT application US 2006/047954, published as WO 2007/075440 and incorporated herein by reference, which describes the work of the applicant herein, discloses a method for assessing the effect of drugs used in antineoplastic therapy in microaggregates of tumor biopsies by first identifying, through labeling with CD31 or through experienced observation, endothelial cells as opposed to neoplastic cells in the sample. The sample, after being subjected to a candidate drug, is stained with a dye which is taken up by dead cells but excluded by viable cells and then counterstained with a dye that is accepted by viable cells. When the sample is examined under a microscope for the particular dye combination selected, the blue/green dye which is excluded by viable cells shows the dead cells as “blueberries” in a background pink “pancake” of viable cells stained with the counterstain. It is possible, therefore, by virtue of the knowledge of which cells are cancer cells and which cells are endothelial cells to determine whether a given drug affects the cancer cells, endothelial cells or both. In general, it is shown that drugs known to inhibit angiogenesis, such as Avastin® (bevacizumab), result in the endothelial cells showing up dead in this assay while the tumor cells remain unaffected. Direct antineoplastic drugs result in the death of the tumor cells themselves, rather than the endothelial cells.
It has now been found that endothelial cells, when caused to become non-viable, not only absorb dyes excluded by viable cells, but also have a distinctive appearance which makes them recognizable even in the presence of other dead cells. Thus, it becomes practical to assess a heterogeneous sample, such as blood plasma, or a disaggregated form or extract of the microaggregates described in WO 2007/075440, for the presence of endothelial cells by subjecting the sample to an agent known to kill endothelial cells and staining with a suitable dye that renders the dead endothelial cells instantly recognizable under a microscope even to an unpracticed eye. This permits a rapid and efficient test for circulating endothelial cells wherein an elevated level of such cells is indicative of traumatic conditions in the subject.
It has also been found, surprisingly, that the nontoxic solvents DMSO and ethanol have themselves antiangiogenic effects and are able to lower the levels of vascular endothelial growth factor (VEGF) in cell cultures.